DNA polymerases are a family of enzymes involved in DNA repair and replication. Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli (Bessman, et al., J. Biol. Chem. 233:171-177 (1957); Buttin and Kornberg J. Biol. Chem. 241:5419-5427 (1966)).
Examples of DNA polymerases isolated from E. coli include E. coli DNA polymerase I and T4 DNA polymerase. These enzymes have a variety of uses in recombinant DNA technology including DNA labeling by nick translation, second-strand cDNA synthesis in cDNA cloning, and DNA sequencing (Maniatis et al., Molecular Cloning: A Laboratory Manual (1982).
U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159 disclosed the use of the above enzymes in a process for amplifying, detecting, and/or cloning nucleic acid sequences. This process, commonly referred to as polymerase chain reaction (PCR), involves the use of a polymerase, primers and nucleotide triphosphates in amplifying existing nucleic acid sequences.
Some of the DNA polymerases discussed above possess a 3'-5' exonuclease activity which provides a proofreading function that gives DNA replication much higher fidelity than it would have if synthesis were the result of only a one base-pairing selection step (Brutlag, D. and Kornberg, A., J. Biol Chem., 247:241-248 (1972)). DNA polymerases with 3'-5' proofreading exonuclease activity have a substantially lower base incorporation error rate when compared with a non-proofreading exonuclease-possessing polymerase (Chang, L. M. S., J. Biol. Chem. 252:1873-1880 (1977)).
Research has also been conducted on the isolation and purification of DNA polymerases from thermophiles, such as Thermus aquaticus. Chien, A. et al. J. Bacteriol. (1976) 127:1550-1557, discloses the isolation and purification of a DNA polymerase with a temperature optimum of 80.degree. C. from T. aquaticus YT1 strain. The Chien et al., purification procedure involves a four-step process. These steps involves preparation of crude extract, DEAE-Sephadex chromatography, phosphocellulose chromatography and chromatography on DNA cellulose.
US Pat. No. 4,889,818 discloses a purified thermostable DNA polymerase from T. aquaticus, Taq polymerase, having a molecular weight of about 86,000 to 90,000 daltons prepared by a process substantially identical to the process of Kaledin with the addition of the substitution of a phosphocellulose chromatography step in lieu of chromatography on single-strand DNA-cellulose. In addition, European Patent Application 0 258 017 disclose Taq polymerase as the preferred enzyme for use in the PCR process discussed above. Research has indicated that while Taq DNA polymerase has a 5'-3' polymerase-dependent exonuclease function, Taq DNA polymerase does not possess a 3'-5' proofreading exonuclease function (Lawyer, et al. J. Biol. Chem. 264:11, p. 6427-6437 (1989). Bernard, et al. Cell 59:219 (1989). As a result, Taq DNA polymerase is prone to base incorporation errors, making its use in certain applications undesirable. For example, attempting to clone an amplified gene is problematic since any one copy of the gene may contain an error due to a random misincorporation event. Depending on where in the replication cycle that error occurs (e.g., in an early replication cycle), the entire DNA amplified could contain the erroneously incorporated base, thus, giving rise to a mutated gene product.
Accordingly, there is a continuing need in the art to obtain and produce a purified, thermostable DNA polymerase with 3'-5' proofreading exonuclease activity that may be used to improve the DNA polymerase processes described above. One such enzyme (describe in more detail below), DNA polymerase I from Thermomicrobium roseum, a Gram negative, pleomorphic, and thermophilic bacterium, has been cloned and expressed in E. coli.